Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.     

Comparative Structural and Functional Characterization of Sorghum and Maize Duplications Containing Orthologous Myb Transcription Regulators of 3-Deoxyflavonoid Biosynthesis

Sequence characterization of the genomic region of sorghum yellow seed 1 shows the presence of two genes that are arranged in a head to tail orientation. The two duplicated gene copies, y1 and y2 are separated by a 9.084 kbp intergenic region, which is largely composed of highly repetitive sequences. The y1 is the functional copy, while the y2 may represent a pseudogene; there are several sequence indels and rearrangements within the putative coding region of y2. The y1 gene encodes a R2R3 type of Myb domain protein that regulates the expression of chalcone synthase, chalcone isomerase and dihydroflavonol reductase genes required for the biosynthesis of 3-deoxyflavonoids. Expression of y1 can be observed throughout the plant and it represents a combination of expression patterns produced by different alleles of the maize p1. Comparative sequence analysis within the coding regions and flanking sequences of y1, y2 and their maize and teosinte orthologs show local rearrangements and insertions that may have created modified regulatory regions. These micro-colinearity modifications possibly are responsible for differential patterns of expression in maize and sorghum floral and vegetative tissues. Phylogenetic analysis indicates that sorghum y1 and y2 sequences may have arisen by gene duplication mechanisms and represent an evolutionarily parallel event to the duplication of maize p2 and p1 genes.     

Characterization of the fungal flora of dolo,a traditional fermented beverage of Burkina Faso,using MALDI-TOF mass spectrometry

A cross-sectional study was conducted in Bobo-Dioulasso, Burkina Faso, to identify the yeast diversity associated with the manufacture of dolo, a traditional fermented beverage of Burkina Faso. From sixty specimens spread onto chromogenic medium plates, sixty-two strains were isolated then identified using MALDI-TOF analysis. Seven yeast species were identified, Saccharomyces cerevisiae (39%) followed by Pichia manshurica (18%) being the most frequent. Forty-three percent of the samples contained Candida species, notably Candida albicans. In conclusion, the combined use of a chromogenic medium and MALDI-TOF analysis reveals a higher diversity in yeast species present in the dolo than previously thought.     

Inactivation of Prions and Amyloid Seeds with Hypochlorous Acid

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥10³- to 10⁶-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~10^5.75-fold and >10⁴-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids.     

The Population Genomics of Trans-Specific Inversion Polymorphisms in Anopheles gambiae

In the malaria mosquito Anopheles gambiae polymorphic chromosomal inversions may play an important role in adaptation to environmental variation. Recently, we used microarray-based divergence mapping combined with targeted resequencing to map nucleotide differentiation between alternative arrangements of the 2La inversion. Here, we applied the same technique to four different polymorphic inversions on the 2R chromosome of An. gambiae. Surprisingly, divergence was much lower between alternative arrangements for all 2R inversions when compared to the 2La inversion. For one of the rearrangements, 2Ru, we successfully mapped a very small region (∼100 kb) of elevated divergence. For the other three rearrangements, we did not identify any regions of significantly high divergence, despite ample independent evidence from natural populations of geographic clines and seasonal cycling, and stable heterotic polymorphisms in laboratory populations. If these inversions are the targets of selection as hypothesized, we suggest that divergence between rearrangements may have escaped detection due to retained ancestral polymorphism in the case of the youngest 2R rearrangements and to extensive gene flux in the older 2R inversion systems that segregate in both An. gambiae and its sibling species An. arabiensis.MORE than 70 years ago Dobzhansky and Sturtevant (1938) first discovered polymorphic inversion arrangements carried by various Drosophila pseudoobscura populations. After observing correlations between environmental conditions and inversion frequencies, Dobzhansky proposed that inversions are under strong selection due to their role in promoting local adaptation to the heterogeneous conditions a species encounters both spatially and temporally (Dobzhansky 1944, 1948; Powell 1997). More recent studies have implicated chromosomal inversions in the adaptation of a diversity of eukaryotes including humans (Coluzzi et al. 1979; Feder et al. 2003; Hoffmann et al. 2004; Stefansson et al. 2005). Long known to be common in dipteran insects, more recent HapMap data suggest that polymorphic inversions may be numerous in human populations and by extension other mammals (Bansal et al. 2007). Given their potential importance in facilitating adaptation, surprisingly little is known about the mechanism(s) or the genes responsible for maintaining inversion polymorphisms in natural populations.Gene exchange between inverted and standard arrangements, although reduced, can still occur through gene flux: the action of gene conversion and multiple crossovers in inversion heterozygotes (heterokaryotypes) (Chovnick 1973; Navarro et al. 1997; Schaeffer and Anderson 2005). Over time allelic variation unrelated to ecological adaptation should become homogenized between arrangements, while alleles which are under divergent selection pressures should remain in linkage disequilibrium with each other and with the inversion itself, leading to heightened differentiation between standard and inverted arrangements at and near the target loci. In principle, this process allows the identification of specific loci involved in adaptive divergence (Schaeffer et al. 2003; Schaeffer and Anderson 2005; Storz 2005). Consistent with this model, previous low-resolution studies of Drosophila inversions revealed heterogeneous patterns of nucleotide diversity relative to divergence, as well as the interspersion of regions of high and low genetic association potentially due to the interaction of selection and gene flux (Schaeffer et al. 2003; Kennington et al. 2006; but see Munte et al. 2005). The application of high-resolution tools flowing from completely sequenced genomes will facilitate the mapping of genes that are the targets of divergent natural selection within gene arrangements.Although Drosophila has been the favored model, the African malaria vector Anopheles gambiae sensu stricto also provides an excellent system for studying the maintenance of inversion polymorphisms, not only within a species but across speciation events of different ages in the An. gambiae sibling species complex. The nominal species An. gambiae s.s. (hereafter, An. gambiae) is synanthropic: almost exclusively biting humans, resting indoors, and exploiting anthropogenic larval habitats (Coluzzi 1999). This close association with humans, vital to making An. gambiae one of the most proficient vectors of malaria, is likely to have been facilitated by chromosomal inversions thought to confer adaptive benefits in heterogeneous climatic and ecological settings in Africa. Seven common polymorphic inversions exist on the second chromosome. Six of these are located on the right arm (2R): j, b, c, u, d, and k, while 2La is the only inversion on the left arm (Coluzzi et al. 2002). Facilitated by the sequenced reference genome (Holt et al. 2002), some of the breakpoints for these polymorphic inversions have been localized to small genomic regions (Sharakhov et al. 2006; Coulibaly et al. 2007; Sangare 2007). Most of these inversions appear to be the targets of strong selection. Five of the inversions (2La and 2Rb, -c, -d, and -u) are nonrandomly associated with degree of aridity; each cycles seasonally with rainfall, and all except 2Ru form stable geographic clines in frequency from mesic forest to xeric regions bordering the Sahara (Coluzzi et al. 1979; Toure et al. 1994, 1998; Powell et al. 1999). Inversion 2Rj is not clinal, but its distribution in Mali is consistent with adaptation to novel rockpool niches (Coluzzi et al. 1985; Manoukis et al. 2008).In the An. gambiae species complex, inversion polymorphisms can be maintained across the boundaries of emerging and even full species. An. gambiae and its sibling An. arabiensis, strictly sympatric throughout most of their extensive ranges in sub-Saharan Africa, differ by multiple fixed chromosomal rearrangements on the X but share three chromosome 2 inversions: 2La, fixed in An. arabiensis and polymorphic in An. gambiae; and 2Rb and -c, polymorphic in both species (Coluzzi et al. 1979, 2002). Moreover, these same inversions and all other common An. gambiae inversions with the exception of 2Rj are shared and polymorphic in two lineages apparently undergoing ecological speciation within An. gambiae—the assortatively mating M and S molecular forms (della Torre et al. 2002, 2005). Inversion frequencies are correlated with climatic and ecological conditions in parallel in both lineages (Costantini et al. 2009; Simard et al. 2009). Unlike the full species, the M and S incipient species are not distinguished by any fixed inversion differences. Indeed, genomewide divergence mapping between the M and S forms revealed that significant differentiation was confined to two small low-recombination regions adjacent to the centromeres of 2L and X which are distant from any inversions (Turner et al. 2005). Thus, in distinction to models of speciation invoking inversions as facilitating the persistence of hybridizing species (Noor et al. 2001; Rieseberg 2001; Ortiz-Barrientos et al. 2002; Navarro and Barton 2003), the An. gambiae data suggest that chromosome 2 inversions are not directly responsible for reproductive isolation. Instead, the same chromosome 2 inversion polymorphisms appear to confer similar ecological benefits, within and across species boundaries. A long-term research goal is to identify the mechanisms and the genes controlling these processes.Previously we conducted the first high-density genomic scan of divergence across a chromosomal inversion (2La) in An. gambiae (White et al. 2007). By hybridizing genomic DNA from S form mosquitoes homokaryotypic for alternate gene arrangements on chromosome 2L (2La or 2L+^a) to oligonucleotide microarrays we were able to measure divergence across the 22-Mb inversion at nearly 14,000 markers. Differentiation in the rearranged region was significantly higher than in collinear portions of chromosome 2L. Between breakpoints the pattern of differentiation was heterogeneous: two genomic clusters of significantly higher divergence were identified near but not adjacent to the breakpoints. Directed resequencing within the S form confirmed these results and suggested that both clusters contained genes targeted by selection. Observed levels of linkage disequilibrium between the 2La breakpoints and markers in the clusters are highly unlikely under a neutral scenario, in light of known recombination rates and plausible estimates of the age of the inversion.The present study characterizes the patterns of genetic variation in polymorphic rearrangements on the opposite (right) arm of chromosome 2: 2Rj, -b, -c, and -u. With the goal of identifying candidate genes maintaining these inversions in natural populations, we applied microarray-based divergence mapping to measure differentiation between alternative 2R arrangements. Because three of four inversions have taxonomic distributions that span incipient and/or completed speciation events, we validated the microarray findings by targeted sequencing in multiple taxa: sympatric Malian populations of An. gambiae M and S forms, and the sibling species An. arabiensis.     

Implication of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in the production of pectinolytic enzymes during cocoa fermentation

The role of bacilli in cocoa fermentation is not well known. Their potential of production of pectinolytic enzymes during this process was evaluated. Bacillus growth was monitored and pectinolytic strains were screened for their use of pectin as sole carbon source. Effects of cocoa fermentation parameters susceptible to influence on enzyme production were analysed. Among 98 strains isolated, 90 were positive for pectin degradation and 80% of them presented detectable pectinolytic activities in submerged fermentation. Forty-eight strains produced polygalacturonase (PG), 47 yielded pectin lyase (PL) and 23 strains produced both enzymes. Bacilli growth was not significantly affected during fermentation. PL production was favoured by galactose, lactose, glucose as sugars, and arginine, glutamine, cysteine and ammonium sulphate as nitrogen compounds. Pectin at low concentration (0.05%) and iron stimulated PL production. It was strongly repressed by galacturonic acid (1%), and negatively affected by nitrogen starvation, zinc and temperatures above 45°C. PL yield was very weak below pH 4.0 and in anaerobic conditions. PG production was weakened by sucrose and cation depletion. It was increased slightly by cysteine, ammonium nitrate and nitrogen starvation and significantly above 40°C. PG synthesis was not affected by acidic pH (3.0–6.0) or oxygen availability. As fermentation products, lactate and acetate lowered the production of both enzymes while ethanol had no effect. The high proportion of pectinolytic producers among the strains studied and analysis of factors influencing pectinolytic enzymes production, suggest that Bacillus sp. is liable to produce at least one enzyme during cocoa fermentation.     

Bimodal targeting of cytochrome P450s to endoplasmic reticulum and mitochondria: the concept of chimeric signals

Targeting signals are critical for proteins to find their specific cellular destination. Signals for protein targeting to the endoplasmic reticulum (ER), mitochondria, peroxisome and nucleus are distinct and the mechanisms of protein translocation across these membrane compartments also vary markedly. Recently, however, a number of proteins have been shown to be present in multiple cellular sites such as mitochondria and ER, cytosol and mitochondria, plasma membrane and mitochondria, and peroxisome and mitochondria suggesting the occurrence of multimodal targeting signals in some cases. Cytochrome P450 monooxygenases (CYPs), which play crucial roles in pharmacokinetics and pharmacodynamics of drugs and toxins, are the prototype of bimodally targeted proteins. Several members of family 1, 2 and 3 CYPs have now been reported to be associated with mitochondria and plasma membrane in addition to the ER. This review highlights the mechanisms of bimodal targeting of CYP1A1, 2B1, 2E1 and 2D6 to mitochondria and ER. The bimodal targeting of these proteins is driven by their N-terminal signals which carry essential elements of both ER targeting and mitochondria targeting signals. These multimodal signals have been termed chimeric signals appropriately to describe their dual targeting property. The cryptic mitochondrial targeting signals of CYP2B1, 2D6, 2E1 require activation by protein kinase A or protein kinase C mediated phosphorylation at sites immediately flanking the targeting signal and/or membrane anchoring regions. The cryptic mitochondria targeting signal of CYP1A1 requires activation by endoproteolytic cleavage by a cytosolic endoprotease, which exposes the mitochondrial signal. This review discusses both mechanisms of bimodal targeting and toxicological consequences of mitochondria targeted CYP proteins.     

Species-specific treatment effects of helminth/HIV-1 co-infection: a systematic review and meta-analysis

In sub-Saharan Africa, over 22 million people are estimated to be co-infected with both helminths and HIV-1. Several studies have suggested that de-worming individuals with HIV-1 may delay HIV-1 disease progression, and that the benefit of de-worming may vary by individual helminth species. We conducted a systematic review and meta-analysis of the published literature to determine the effect of treatment of individual helminth infections on markers of HIV-1 progression (CD4 count and HIV viral load). There was a trend towards an association between treatment for Schistosoma mansoni and a decrease in HIV viral load (Weighted mean difference (WMD)=-0·10; 95% Confidence interval (CI): -0·24, 0·03), although this association was not seen for Ascaris lumbricoides, hookworm or Trichuris trichiura. Treatment of A. lumbricoides, S. mansoni, hookworm or T. trichiura was not associated with a change in CD4 count. While pooled data from randomized trials suggested clinical benefit of de-worming for individual helminth species, these effects decreased when observational data were included in the pooled analysis. While further trials are needed to confirm the role of anthelmintic treatment in HIV-1 co-infected individuals, providing anthelmintics to individuals with HIV-1 may be a safe, inexpensive and practical intervention to slow progression of HIV-1.